Karin E. Bakkelund

Gut luminal microdialysis of glycerol as a marker of intestinal ischemic injury and recovery

Objective:To evaluate microdialysis as a method to assess different degrees of intestinal damage and recovery during ischemia and reperfusion; to evaluate information obtained from microdialysis catheters in the peritoneum, the gut wall, and the gut lumen. Design:Randomized, controlled animal experiment. Setting:University laboratory animal center. Subjects:Twenty-seven domestic pigs. Interventions:The superior mesenteric artery was cross-clamped for 60 mins (n = 14) or 120 mins (n = 10) followed by 2 or 4 hrs of reperfusion. Three pigs served as controls. Measurements and Main Results:Intestinal mucosal integrity was assessed by morphometry, adenosine triphosphate in the gut wall, and permeability of 14C-polyethylene glycol. Lactate, glycerol, pyruvate, and glucose were measured by microdialysis. Changes in adenosine triphosphate, permeability, or lactate did not correlate to different extents of intestinal damage caused by 60 or 120 mins of ischemia. During the reperfusion period, pigs with 60 mins of intestinal ischemia showed a faster recovery of these variables than pigs with 120 mins of intestinal ischemia. Glycerol increased with increasing duration of the ischemic insult. After 60 mins of intestinal ischemia, glycerol in the gut lumen decreased toward baseline but remained high after 120 mins of intestinal ischemia. There was a good correlation between gut luminal glycerol and recovery of mucosal damage throughout the reperfusion period. In the peritoneal cavity, both glycerol and lactate decreased to baseline relatively shortly after onset of reperfusion independent of the duration of intestinal ischemia. Conclusions:Microdialysis of glycerol provides information about the extent and severity of intestinal damage after ischemia and about the ensuing recovery. The gut lumen is to be preferred as a site for placement of microdialysis catheters.

Signet Ring Cells in Gastric Carcinomas Are Derived from Neuroendocrine Cells

Adenocarcinomas are malignant tumors with glandular growth and/or supposed intracellular mucin as identified by periodic acid-Schiff (PAS) positivity. Gastric signet ring cell carcinomas are classified as diffuse type. A proportion of diffuse-type adenocarcinomas have previously been suggested to be of neuroendocrine origin. In the present study we examined gastric signet ring cell carcinomas for neuroendocrine differentiation. Of 11 gastric signet ring cell carcinomas, 8 contained areas with PAS-positive signet ring cells that also were immunoreactive for one or several neuroendocrine markers: synaptophysin, chromogranin A, and histidine decarboxylase, the latter an enterochromaffin-like (ECL) cell marker. Whereas PAS positivity was located in the central cytoplasm, neuroendocrine immunoreactivity was often located as a rim surrounding an otherwise non-immunoreactive cytoplasm, presumed to represent the area with PAS-positive material. These findings indicate that signet ring cell carcinomas could be of neuroendocrine origin. We propose that signet ring cell carcinomas develop by gradual dedifferentiation from ECL cells via signet ring cells with neuroendocrine immunoreactivity toward signet ring cells where the cytoplasm mainly consists of PAS-positive material. This finding could have implications for the classification and understanding of gastric carcinogenesis.

ECL-Cell Derived Gastric Cancer in Male Cotton Rats Dosed with the H2-Blocker Loxtidine

Spontaneously hypergastrinemic cotton rats (Sigmodon hispidus) develop tumors that have the phenotype of an adenocarcinoma but most likely originate from the enterochromaffin-like (ECL) cells. Among inbred animals ∼50% of the females, but <1% of males develop spontaneous gastric carcinomas. Gastrin is the principle carcinogen in this model, as >4 months of hypergastrinemia results in carcinoma, but a gastrin receptor antagonist prevents carcinomas. Carcinomas can also be induced by partial corpectomy. In the present study, the insurmountable H2-receptor antagonist loxtidine (200 mg/kg/day) was given to male cotton rats for 6 months. The loxtidine-dosed animals developed hypergastrinemia, whereas control animals remained normogastrinemic. At termination, 4 of 5 cotton rats had cancer located to the oxyntic mucosa, whereas 1 animal had dysplasia. The gastric mucosa of all of the control animals was normal. In the dysplastic mucosa of loxtidine-dosed animals there was a marked increase in chromogranin A-positive cells, where numerous groups of cells also stained positive with the Sevier-Munger technique. In areas of high proliferation and cancer there were also histidine decarboxylase, chromogranin A, and Sevier-Munger-positive cells, altogether indicating an ECL cell origin of the tumors. This represents an interesting animal model where ECL cell-derived gastric cancer can be induced by pharmacological acid inhibition in 6 months.

Hypergastrinaemia induced by partial corpectomy results in development of enterochromaffin-like cell carcinoma in male Japanese cotton rats.

Background: Among inbred female cotton rats (Sigmodon hispidus) 25%–50% of the animals develop spontaneous gastric carcinomas, whereas males have an incidence of less than 1%. The carcinomas are enterochromaffin‐like (ECL)‐cell derived. Animals with gastric carcinomas also have hypergastrinaemia and gastric hypoacidity, but the mechanism behind the hypoacidity is unknown. Carcinomas have been found in all female cotton rats with spontaneous hypergastrinaemia lasting more than 4 months, and a gastrin receptor antagonist prevents the development of carcinoma. The purpose of the present study was to investigate whether induced hypergastrinaemia in male cotton rats would also result in carcinomas. Methods: Hypergastrinaemia was induced by partial corpectomy of male cotton rats, aiming at removal of 80%–90% of the corpus. A control group was sham‐operated. Results: All partially corpectomized animals developed persistent hypergastrinaemia. Six months after the operation, 9 out of 13 partially corpectomized animals developed gastric cancer. In the dysplastic mucosa surrounding the tumours there was an increase in chromogranin A immunoreactive cells, where numerous cells also were stained using the Sevier‐Munger technique. Tumour tissue also contained cells that were chromogranin A positive and stained by Sevier‐Munger. Conclusions: ECL‐cell carcinomas can be induced in male cotton rats by partial corpectomy. This supports a previous statement that spontaneous carcinomas in female cotton rats are caused by gastric hypoacidity and hypergastrinaemia. In hypergastrinaemic animals, ECL‐cell carcinomas develop independently of gender within a relatively short period of time, and cotton rats therefore represent an interesting model for studying gastric carcinogenesis.

Long-term gastric changes in achlorhydric H+/K+-ATPase beta subunit deficient mice

Abstract Objective. Hypergastrinemia is known to induce enterochromaffin-like (ECL) cell derived tumors in rodents and man. In this study, we have examined the effect of life-long gastric anacidity and secondary hypergastrinemia in H+/K+-ATPase beta subunit knockout (KO) mice. Material and methods. Female H+/K+-ATPase beta subunit KO mice and controls were followed up to 20 months before being sacrificed. At termination, intragastric acidity was measured and internal organs were examined for macroscopic and histological changes. Plasma gastrin and serum albumin were measured. Results. KO mice were anacidic and hypergastrinemic. The oxyntic mucosa was markedly, and with increase in age, hyperplastic with cystic dilatations resembling the changes seen in patients with Menetriers disease. Serum albumin in KO mice did not differ from controls. KO mice had a marked ECL cell hyperplasia, but only one gastric carcinoma was found. Conclusion. H+/K+-ATPase beta subunit KO mice develop Menetrier-like changes in the stomach, and may be useful in studying the pathogenesis and treatment of Menetriers disease. The reason why only one KO mice developed gastric neoplasia whereas the histamine-2 blocker loxtidine has previously been found to regularly induce ECL cell carcinoids in mice is not known.

Neuroendocrine cells in diffuse gastric carcinomas: an ultrastructural study with immunogold labeling of chromogranin A.

Neuroendocrine differentiation is often found in gastric carcinomas, but the relevance of these cells in gastric carcinogenesis is debated. We applied immunolabeling at the electron microscopic level to study the ultrastructure of neuroendocrine cells in gastric carcinomas to ensure correct cellular classification of dedifferentiated cells. The immunogold labeling at electron microscopic level was compared with an established sensitive immunohistochemical method using light microscopy. Thirteen human gastric adenocarcinomas of the diffuse type were examined for neuroendocrine differentiation by chromogranin A (CgA) labeling at both the light and electron microscopic level. The ultrastructure of CgA-positive cells was compared with CgA-positive cells from controls. Nine of 13 tumors showed CgA-positive cells both at the light and electron microscopic level. The CgA-positive cells displayed altered ultrastructural features compared with controls. Some of the CgA-positive tumor cells had granules typical for enterochromaffin-like cells. Immunoelectron microscopy seems to provide both significant immunolabeling and sufficient ultrastructure to enhance classification of cells in neoplastic tissue.

Role of bradykinin in gastric vasodilation caused by hypertonic saline and acid back diffusion

Protective vasodilation in response to tissue injury and acid back diffusion is associated with release of bradykinin in the rat stomach. We hypothesized that bradykinin might be involved in mechanisms behind such vasodilation via influence on mast cells and sensory neurons. Acid back diffusion after mucosal barrier disruption with hypertonic saline evoked degranulation of mast cells in the rat stomach wall. Acid back diffusion was also associated with increased luminal release of histamine and gastric blood flow in normal rats, but not in mast cell-deficient rats. Bradykinin (BK(2)) receptor blockade inhibited degranulation of submucosal mast cells in the stomach and attenuated gastric vasodilation both in response to acid back diffusion and after stimulation of sensory neurons with capsaicin. Gastric vasodilation caused by mucosal injury with hypertonic saline alone was associated with degranulation of mucosal mast cells. These events were unaffected by inhibition of prostaglandin synthesis, whereas bradykinin (BK(2)) receptor blockade was associated with abolished vasodilation and inhibition of mucosal mast cell degranulation. We conclude that bradykinin is involved in gastric vasodilation caused by hypertonic injury alone via influence on mast cells, and by acid back diffusion via influence on both sensory neurons and mast cells.

Gastric carcinomas localized to the cardia.

Objectives. Gastric adenocarcinomas localized to the cardia are increasing. Enterochromaffin-like (ECL) cells play a role in gastric carcinogenesis in hypergastrinemia, and the use of proton pump inhibitors (PPI) leading to hypergastrinemia has increased considerably during the last decades. We have examined cardia cancers for neuroendocrine and ECL cell differentiation. Methods. Thirty-two cardia cancers were examined by immunohistochemical labelling of chromogranin A (CgA), synaptophysin, serotonin, and histidine decarboxylase (HDC). Information about PPI use was collected from the patient records. Results. In 15 of 32 tumours, there were positive signs for one or several neuroendocrine markers. Five cases were CgA and serotonin positive; three of these carcinomas were also positive for HDC. Three patients were long-term users of PPI, and two of these were immunoreactive for neuroendocrine markers. Conclusions. A high proportion of cardia cancers expressed neuroendocrine markers, but only few patients with cardia cancers were using PPI.

Hemodynamic and tissue blood flow responses to long-term pneumoperitoneum and hypercapnia in the pig

BackgroundIncreased peritoneal blood flow may influence the ability of cancer cells to adhere to and survive on the peritoneal surface during and after laparoscopic cancer surgery. Carbon dioxide (CO2) pneumoperitoneum is associated with a marked blood flow increase in the peritoneum. However, it is not clear whether the vasodilatory effect in the peritoneum is related to a local or systemic effect of CO2.MethodsIn this study, 21 pigs were exposed to pneumoperitoneum produced with either CO2 (n = 7) or helium (He) (n = 7) insufflation at 10 mmHg for 4 h, or to two consecutive levels of hypercapnia (7 and 11 kPa) (n = 7) produced by the addition of CO2 to the inhalational gas mixture. Tissue blood flow measurements were performed using the colored microsphere technique.ResultsBlood flow in peritoneal tissue increased during CO2, but not He, pneumoperitoneum, whereas it did not change at any level of hypercapnia alone. There was no change in blood flow in most organs at the partial pressure of CO2 (PaCO2) level of 7 kPa. However, at a PaCO2 of 11 kPa, blood flow was increased in the central nervous system, myocardium, and some gastrointestinal organs. The blood flow decreased markedly in all striated muscular tissues during both levels of hypercapnia.ConclusionThe effect of CO2 on peritoneal blood flow during laparoscopic surgery is a local effect, and not attributable to central hemodynamic effects of CO2 pneumoperitoneum or high systemic levels of CO2.

The effects of unilateral truncal vagotomy on gastric carcinogenesis in hypergastrinemic Japanese female cotton rats.

The stomach is innervated by the vagal nerve. Several studies have demonstrated that the vagal nerve has a trophic effect on the rat oxyntic mucosa and that the trophic effect of hypergastrinemia is dependent on intact vagal innervation. The effect of vagal denervation on gastric carcinogenesis has been examined in Mastomys natalensis and hypergastrinemic transgenic INS-GAS mice, with no effect of unilateral vagotomy in Mastomys but an anti-carcinogenic effect in INS-GAS mice. A proportion of female Japanese cotton rats develop spontaneous hypergastrinemia and ECL cell derived gastric carcinomas. In the current study we have examined the effects of unilateral anterior subdiaphragmatic vagotomy on gastric carcinogenesis. Female Japanese cotton rats were operated with unilateral anterior vagotomy or sham-operation at age 2 months and were terminated at age 10 months. Ten of fifteen animals operated by anterior vagotomy and 11 of 16 sham-operated developed hypergastrinemia. Vagotomy did not affect intragastric pH or serum gastrin. When comparing the anterior and posterior sides of the stomachs, vagotomy did not affect the occurrence of dysplasia or carcinoma development in the oxyntic mucosa. However, vagotomy resulted in lower stomach weight and reduced oxyntic mucosal thickness on the anterior side. Vagotomy also resulted in a reduction in volume density of chromogranin A positive cells in the oxyntic mucosa. In conclusion, vagotomy reduced the trophic effects of hypergastrinemia on the ECL cell and oxyntic mucosa, but did not prevent gastric carcinogenesis in female Japanese cotton rats. The effects of vagotomy on gastric carcinogenesis in animal models are conflicting and further studies in patients should be done to clarify the clinically significant effects of vagotomy.